The Developmentally Regulated Osteoblast Phosphodiesterase GDE3 Is Glycerophosphoinositol-specific and Modulates Cell Growth
2009; Elsevier BV; Volume: 284; Issue: 37 Linguagem: Inglês
10.1074/jbc.m109.035444
ISSN1083-351X
AutoresDaniela Corda, Takahiro Kudo, Pasquale Zizza, Cristiano Iurisci, Eri Kawai, Norihisa Katô, Noriyuki Yanaka, Stefania Mariggiò,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoThe glycerophosphodiester phosphodiesterase enzyme family involved in the hydrolysis of glycerophosphodiesters has been characterized in bacteria and recently identified in mammals. Here, we have characterized the activity and function of GDE3, one of the seven mammalian enzymes. GDE3 is up-regulated during osteoblast differentiation and can affect cell morphology. We show that GDE3 is a glycerophosphoinositol (GroPIns) phosphodiesterase that hydrolyzes GroPIns, producing inositol 1-phosphate and glycerol, and thus suggesting specific roles for this enzyme in GroPIns metabolism. Substrate specificity analyses show that wild-type GDE3 selectively hydrolyzes GroPIns over glycerophosphocholine, glycerophosphoethanolamine, and glycerophosphoserine. A single point mutation in the catalytic domain of GDE3 (GDE3R231A) leads to loss of GroPIns enzymatic hydrolysis, identifying an arginine residue crucial for GDE3 activity. After heterologous GDE3 expression in HEK293T cells, phosphodiesterase activity is detected in the extracellular medium, with no effect on the intracellular GroPIns pool. Together with the millimolar concentrations of calcium required for GDE3 activity, this predicts an enzyme topology with an extracellular catalytic domain. Interestingly, GDE3 ectocellular activity is detected in a stable clone from a murine osteoblast cell line, further confirming the activity of GDE3 in a more physiological context. Finally, overexpression of wild-type GDE3 in osteoblasts promotes disassembly of actin stress fibers, decrease in growth rate, and increase in alkaline phosphatase activity and calcium content, indicating a role for GDE3 in induction of differentiation. Thus, we have identified the GDE3 substrate GroPIns as a candidate mediator for osteoblast proliferation, in line with the GroPIns activity observed previously in epithelial cells. The glycerophosphodiester phosphodiesterase enzyme family involved in the hydrolysis of glycerophosphodiesters has been characterized in bacteria and recently identified in mammals. Here, we have characterized the activity and function of GDE3, one of the seven mammalian enzymes. GDE3 is up-regulated during osteoblast differentiation and can affect cell morphology. We show that GDE3 is a glycerophosphoinositol (GroPIns) phosphodiesterase that hydrolyzes GroPIns, producing inositol 1-phosphate and glycerol, and thus suggesting specific roles for this enzyme in GroPIns metabolism. Substrate specificity analyses show that wild-type GDE3 selectively hydrolyzes GroPIns over glycerophosphocholine, glycerophosphoethanolamine, and glycerophosphoserine. A single point mutation in the catalytic domain of GDE3 (GDE3R231A) leads to loss of GroPIns enzymatic hydrolysis, identifying an arginine residue crucial for GDE3 activity. After heterologous GDE3 expression in HEK293T cells, phosphodiesterase activity is detected in the extracellular medium, with no effect on the intracellular GroPIns pool. Together with the millimolar concentrations of calcium required for GDE3 activity, this predicts an enzyme topology with an extracellular catalytic domain. Interestingly, GDE3 ectocellular activity is detected in a stable clone from a murine osteoblast cell line, further confirming the activity of GDE3 in a more physiological context. Finally, overexpression of wild-type GDE3 in osteoblasts promotes disassembly of actin stress fibers, decrease in growth rate, and increase in alkaline phosphatase activity and calcium content, indicating a role for GDE3 in induction of differentiation. Thus, we have identified the GDE3 substrate GroPIns as a candidate mediator for osteoblast proliferation, in line with the GroPIns activity observed previously in epithelial cells. The glycerophosphodiester phosphodiesterases (GP-PDEs) 5The abbreviations used are:GP-PDEglycerophosphodiester phosphodiesteraseRGS16regulator of G-protein signaling 16GPIsglycerophosphoinositolsGroPInsglycerophosphoinositolGroPIns4Pglycerophosphoinositol 4-phosphatePLA2IVαphospholipase A2 type IVαGroPChoglycerophosphocholineIns1Pinositol 1-phosphateCHO cellsChinese hamster ovary cellsHEK293Thuman embryonic kidney 293T cellsHPLChigh performance liquid chromatographyFCSfetal calf serumPBSphosphate-buffered salineGFPgreen fluorescent proteinwtwild type. were initially characterized in bacteria, where they have functional roles for production of metabolic carbon and phosphate sources from glycerophosphodiesters (1Larson T.J. Ehrmann M. Boos W. J. Biol. Chem. 1983; 258: 5428-5432Abstract Full Text PDF PubMed Google Scholar, 2Tommassen J. Eiglmeier K. Cole S.T. Overduin P. Larson T.J. Boos W. Mol. Gen. Genet. 1991; 226: 321-327Crossref PubMed Scopus (71) Google Scholar) and in adherence to and degradation of mammalian host-cell membranes (3Ahrén I.L. Janson H. Forsgren A. Riesbeck K. Microb. Pathog. 2001; 31: 151-158Crossref PubMed Scopus (43) Google Scholar). The GP-PDEs have a catalytic region of 56 amino acids (4Zheng B. Berrie C.P. Corda D. Farquhar M.G. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 1745-1750Crossref PubMed Scopus (69) Google Scholar). After their characterization in bacteria, mammalian glycerophosphodiesterases were identified, with the definition of a family of seven members (5Yanaka N. Biosci. Biotechnol. Biochem. 2007; 71: 1811-1818Crossref PubMed Scopus (52) Google Scholar). The first of these, GDE1, is an interactor of regulator of G-protein signaling (RGS)16, and was subsequently defined as a GP-PDE regulated by G-protein signaling (4Zheng B. Berrie C.P. Corda D. Farquhar M.G. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 1745-1750Crossref PubMed Scopus (69) Google Scholar). Indeed, GDE1 expression in HEK293T cells showed increased enzymatic activity upon α/β-adrenergic and lysophospholipid receptor stimulation (4Zheng B. Berrie C.P. Corda D. Farquhar M.G. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 1745-1750Crossref PubMed Scopus (69) Google Scholar). The second member, GDE2, was isolated by homology searches in neuronal tissues and its physiological role involves neuronal differentiation (6Rao M. Sockanathan S. Science. 2005; 309: 2212-2215Crossref PubMed Scopus (69) Google Scholar, 7Yanaka N. Nogusa Y. Fujioka Y. Yamashita Y. Kato N. FEBS Lett. 2007; 581: 712-718Crossref PubMed Scopus (25) Google Scholar). In contrast, GDE3 has been characterized as a marker of osteoblast differentiation and was isolated through a differential display method (8Yanaka N. Imai Y. Kawai E. Akatsuka H. Wakimoto K. Nogusa Y. Kato N. Chiba H. Kotani E. Omori K. Sakurai N. J. Biol. Chem. 2003; 278: 43595-43602Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). GDE4 was isolated only recently with three-dimensional modeling defining it as a GP-PDE, although no functional activity has been correlated to its expression (9Chang P.A. Shao H.B. Long D.X. Sun Q. Wu Y.J. Mol. Membr. Biol. 2008; 25: 557-566Crossref PubMed Scopus (11) Google Scholar). The remaining members were cloned following data base searches, with further studies required for the definition of their properties (5Yanaka N. Biosci. Biotechnol. Biochem. 2007; 71: 1811-1818Crossref PubMed Scopus (52) Google Scholar). The diversity among these family members, in terms of tissue distribution, subcellular localization, and substrate specificity, suggests they selectively regulate biological functions and have distinct physiological roles (5Yanaka N. Biosci. Biotechnol. Biochem. 2007; 71: 1811-1818Crossref PubMed Scopus (52) Google Scholar).The only GP-PDE activity that has been biochemically characterized to date followed GDE1 overexpression in HEK293T cells, which showed a selectivity for the glycerophosphoinositols (GPIs) as substrate (4Zheng B. Berrie C.P. Corda D. Farquhar M.G. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 1745-1750Crossref PubMed Scopus (69) Google Scholar), in contrast to the bacterial GP-PDEs that show broad substrate specificities with respect to the alcohol moiety of the glycerophosphodiesterases (1Larson T.J. Ehrmann M. Boos W. J. Biol. Chem. 1983; 258: 5428-5432Abstract Full Text PDF PubMed Google Scholar, 2Tommassen J. Eiglmeier K. Cole S.T. Overduin P. Larson T.J. Boos W. Mol. Gen. Genet. 1991; 226: 321-327Crossref PubMed Scopus (71) Google Scholar). The GPIs are naturally occurring, biologically active metabolites of the phosphoinositides that were originally investigated in the context of Ras-transformed cells (10Valitutti S. Cucchi P. Colletta G. Di Filippo C. Corda D. Cell. Signal. 1991; 3: 321-332Crossref PubMed Scopus (38) Google Scholar). They are present in virtually all cell types, where their intracellular levels can also be modulated according to cell activation, differentiation, and development (Refs. 11Corda D. Iurisci C. Berrie C.P. Biochim. Biophys. Acta. 2002; 1582: 52-69Crossref PubMed Scopus (75) Google Scholar and 12Corda D. Zizza P. Varone A. Filippi B.M. Mariggiò S. Cell. Mol. Life Sci. 2009; PubMed Google Scholar and references therein). Recently, glycerophosphoinositol (GroPIns) was characterized as a mediator of purinergic and adrenergic regulation of PCCl3 thyroid cell proliferation (13Mariggiò S. Sebastià J. Filippi B.M. Iurisci C. Volontè C. Amadio S. De Falco V. Santoro M. Corda D. Faseb J. 2006; 20: 2567-2569Crossref PubMed Scopus (27) Google Scholar), while GroPIns 4-phosphate (GroPIns4P) has been shown to induce reorganization of the actin cytoskeleton in fibroblasts and in T-lymphocytes, by promoting a sustained and robust activation of the Rho GTPases (14Mancini R. Piccolo E. Mariggiò S. Filippi B.M. Iurisci C. Pertile P. Berrie C.P. Corda D. Mol. Biol. Cell. 2003; 14: 503-515Crossref PubMed Scopus (23) Google Scholar, 15Filippi B.M. Mariggiò S. Pulvirenti T. Corda D. Biochim. Biophys. Acta. 2008; 1783: 2311-2322Crossref PubMed Scopus (16) Google Scholar, 16Patrussi L. Mariggiò S. Paccani S.R. Capitani N. Zizza P. Corda D. Baldari C.T. Cell. Signal. 2007; 19: 2351-2360Crossref PubMed Scopus (12) Google Scholar).The GPIs appear to rapidly equilibrate across the plasma membrane when added exogenously to cells, to exert their actions within the cell (12Corda D. Zizza P. Varone A. Filippi B.M. Mariggiò S. Cell. Mol. Life Sci. 2009; PubMed Google Scholar). The plasma membrane transporter for GroPIns characterized in yeast is the protein GIT1 (17Patton-Vogt J.L. Henry S.A. Genetics. 1998; 149: 1707-1715Crossref PubMed Google Scholar), with one of its orthologs in mammalian cells identified as the human permease Glut2 (18Mariggiò S. Iurisci C. Sebastià J. Patton-Vogt J. Corda D. FEBS Lett. 2006; 580: 6789-6796Crossref PubMed Scopus (15) Google Scholar). This specific transporter has been proposed to mediate both GroPIns uptake and release, which depends on the GroPIns concentration gradient across the plasma membrane. Under physiological conditions, this gradient can arise from the formation of GPIs from the phosphoinositides inside cells following activation of a specific isoform of phospholipase A2, PLA2IVα (13Mariggiò S. Sebastià J. Filippi B.M. Iurisci C. Volontè C. Amadio S. De Falco V. Santoro M. Corda D. Faseb J. 2006; 20: 2567-2569Crossref PubMed Scopus (27) Google Scholar, 19Mariggiò S. Filippi B.M. Iurisci C. Dragani L.K. De Falco V. Santoro M. Corda D. Cancer Res. 2007; 67: 11769-11778Crossref PubMed Scopus (13) Google Scholar).The release of the GPIs into the extracellular medium can affect their paracrine targets (16Patrussi L. Mariggiò S. Paccani S.R. Capitani N. Zizza P. Corda D. Baldari C.T. Cell. Signal. 2007; 19: 2351-2360Crossref PubMed Scopus (12) Google Scholar) or initiate their catabolism. This is supported by our characterization of GDE1 activity, and now of GDE3 activity, both of which show a substrate selectivity toward GroPIns, and catalytic activity after heterologous expression that can only be monitored in the extracellular space. Interestingly, GDE3 activity appears to be related to modulation of osteoblast functions, delineating a role for GDE3 in promoting osteoblast differentiation, and mainly regulating osteoblast proliferation.EXPERIMENTAL PROCEDURESMaterialsDulbecco's modified Eagle's medium (DMEM), Minimal Essential Medium α (MEMα), fetal calf serum (FCS), Opti-MEM, phosphate-buffered saline (PBS), bovine serum albumin, and Hank's Balanced Salt Solution with calcium and magnesium (HBSS++) were from Invitrogen Brl (Grand Island, NY). [3H]GroPIns was prepared from l-α-[3H]phosphatidylinositol (314.5 GBq/mmol; PerkinElmer, Boston, MA) by deacylation, according to the original procedure of Clarke and Dawson (20Clarke N.G. Dawson R.M. Biochem. J. 1981; 195: 301-306Crossref PubMed Scopus (185) Google Scholar). [6-3H]Thymidine (18.4 Ci/mmol) was from PerkinElmer. GroPIns was purchased from Euticals S.p.A. (Lodi, Italy) as its calcium salt, and from Calbiochem (La Jolla, CA) as its lithium salt. All other reagents were of the highest purities available and were obtained from Sigma, unless otherwise specified.Plasmids, Cell Culture, Transfection, and Proliferation AssaysFull-length mouse GDE3 cDNA was subcloned into the expression vector pCMV-EGFPN1 (pEGFP-GDE3wt), as reported previously (8Yanaka N. Imai Y. Kawai E. Akatsuka H. Wakimoto K. Nogusa Y. Kato N. Chiba H. Kotani E. Omori K. Sakurai N. J. Biol. Chem. 2003; 278: 43595-43602Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). An Arg→Ala mutant (pEGFP-GDE3R231A) was produced using the QuikChangeTM site-directed mutagenesis kit (Stratagene), a set of PCR primers (5′-CAGCATGGGGGCCCCTGCGTGTCCCACCAGCCC-3′ and 5′-GGGCTGGTGGGACACGCAGGGGCCCCCATGCTG-3′), and pEGFP-GDE3 as a template, according to the manufacturer's protocol. The mutation was verified by DNA sequencing using an ABI PRISM 310 Genetic analyzer (Applied Biosystems).HEK293T cells (American Type Culture Collection) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% FCS, 2 mm l-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin, in a humidified atmosphere of 5% CO2 in air, at 37 °C. Chinese hamster ovary (CHO) cells were maintained in DMEM supplemented with 10% FCS, 58 μg/ml proline, 53 μg/ml l-aspartic acid, 60 μg/ml l-asparagine, 100 units/ml penicillin, and 100 μg/ml streptomycin. HEK293T and CHO cells were transiently transfected with 4 μg of pEGFP-GDE3wt, pEGFP-GDE3R231A, or vector pEGFP (control) using Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions.Stable clones of the MC3T3-E1 murine osteoblastic cell line (Dainippon Pharmaceutical Co., Osaka, Japan) were transfected with the empty vector (pEF/neoI) or with pEF-GDE3 (MC3T3-E1-Cl15), and were prepared as reported previously (8Yanaka N. Imai Y. Kawai E. Akatsuka H. Wakimoto K. Nogusa Y. Kato N. Chiba H. Kotani E. Omori K. Sakurai N. J. Biol. Chem. 2003; 278: 43595-43602Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The cells were maintained in MEMα supplemented with 10% FCS, 2 mm l-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin, in a humidified atmosphere of 5% CO2 in air at 37 °C, in the presence of the selection antibiotic G418 (500 μg/ml).The osteoblast growth rate was evaluated by cell counting. The different clones were plated into 6-well plates (2 × 104 cell/well) in growth medium. At the indicated times, the cells were detached by trypsinization, recovered by centrifugation, and put through two independent and blinded cell counts (Neubauer cell-counting chamber).For the [3H]thymidine incorporation assay, MC3T3-E1 and MC3T3-E1-Cl15 cells were seeded in 96-well plates at a density of 5 × 103 cell/well in complete growth medium. After 12 h, the cells were treated with 250 μm GroPIns (calcium salt) or the equimolar 125 μm CaCl2 and, when indicated, two further additions followed after 36 and 60 h. After 72 h, a pulse of [3H]thymidine (1 μCi/well) was given 4 h before stopping the reaction by washing twice with HBSS++. The [3H]thymidine incorporation into trichloroacetic acid-insoluble material was evaluated as previously described (13Mariggiò S. Sebastià J. Filippi B.M. Iurisci C. Volontè C. Amadio S. De Falco V. Santoro M. Corda D. Faseb J. 2006; 20: 2567-2569Crossref PubMed Scopus (27) Google Scholar, 21Sowa H. Kaji H. Yamaguchi T. Sugimoto T. Chihara K. J Bone Miner Res. 2002; 17: 1190-1199Crossref PubMed Scopus (85) Google Scholar).Postnuclear Lysate Preparation and Western BlottingTwenty-four hours after transfection, cells were washed twice with cold PBS and scraped into homogenization buffer containing protease inhibitors (0.5 μg/ml leupeptin, 2 μg/ml aprotinin, 0.5 mm phenanthroline, 2 μm pepstatin, and 1 mm phenylmethylsulfonyl fluoride), and 5 mm EDTA in TBS (20 mm Tris-HCl, pH 7.5, 500 mm NaCl). Following gentle homogenization by 10 passages through a 28 1/2-gauge needle, postnuclear supernatants were prepared by removing nuclei and unbroken cells by centrifugation (600 × g for 3 min, at 4 °C) according to Ref. 4Zheng B. Berrie C.P. Corda D. Farquhar M.G. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 1745-1750Crossref PubMed Scopus (69) Google Scholar. Eighty micrograms of supernatant protein was subjected to SDS-PAGE, and Western blotting was performed with a polyclonal anti-GFP antibody (a kind gift of G. Di Tullio, Consorzio Mario Negri Sud, Italy) and a polyclonal antibody against GDE3 (epitope: amino acids 210–332) (8Yanaka N. Imai Y. Kawai E. Akatsuka H. Wakimoto K. Nogusa Y. Kato N. Chiba H. Kotani E. Omori K. Sakurai N. J. Biol. Chem. 2003; 278: 43595-43602Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Western blots were developed using the chemiluminescent method (ECL, Amersham Biosciences).GP-PDE Activity AssaysIncubations were routinely at 37 °C with postnuclear supernatants in a final volume of 50 μl, which included 100 mm Tris-HCl, pH 7.5, 10 mm MgCl2, 2 mg/ml fatty-acid-free bovine serum albumin, 30,000 dpm [3H]GroPIns, [3H]GroPIns4P, or [3H]glycerophosphocholine (GroPCho), unlabeled GroPIns, GroPIns4P, or GroPCho (as indicated), without or with addition of competing glycerophosphodiesters. Incubations were in the presence of 5 mm Ca2+ or as otherwise specified. Initial analysis of incubation conditions for the GP-PDE assay indicated that with 10 mm GroPIns as substrate, increasing amounts of postnuclear protein preparations (2, 10, 30 μg) from HEK293T cells overexpressing GFP-GDE3wt maintained linear activities in a 2-h incubation at 37 °C. Thus, 10 μg of postnuclear protein preparations were routinely used, unless otherwise specified. The GroPIns dose-response analysis was performed using 5 mm Ca2+, GroPIns substrate as the Li+ salt (from 100 μm to 500 mm), 10 μg of postnuclear lysate protein, and a 2-h incubation at 37 °C. GroPIns hydrolysis was determined as that measured for GFP-GDE3wt over the background GFP postnuclear preparation control, expressed as nanomoles. In the GP-PDE competition assays, the substrate concentration was 1 mm GroPIns, with addition of a 10-fold excess (10 mm) of competing glycerophosphodiesters.For the in vivo extracellular GP-PDE assays, cells were plated in 6-well plates and 100,000 dpm/well [3H]GroPIns, [3H]GroPIns4P, or [3H]GroPCho were added in 2.5 ml of growth medium. At specific times, 500 μl of medium was analyzed, as indicated below. The incubations were terminated by addition of cold methanol (−20 °C), followed by two-phase extraction, and the lyophilizing of the resultant upper (aqueous) phase (further details in Ref. 22Berrie C.P. Iurisci C. Piccolo E. Bagnati R. Corda D. Methods Enzymol. 2007; 434: 187-232Crossref PubMed Scopus (16) Google Scholar). For the dose-response curves, GP-PDE activities were calculated from the known cold GroPIns in each assay (pmol) and the level of postincubation GroPIns hydrolysis, as seen by HPLC analysis of the 3H-labeled inositol, inositol 1-phosphate (Ins1P), and GroPIns (further details in Ref. 22Berrie C.P. Iurisci C. Piccolo E. Bagnati R. Corda D. Methods Enzymol. 2007; 434: 187-232Crossref PubMed Scopus (16) Google Scholar).Quantitative Cell Spreading Assay and Immunofluorescence AnalysisHEK293T cells were transiently transfected with pEGFP-GDE3wt, pEGFP-GDE3R231A, or pEGFP vector (control), as described above. After 24 h, the cells were directly fixed with 4% paraformaldehyde and 4% sucrose in 0.2 m NaPO4 (pH 7.2), for 30 min. At least three images from different regions of the dish were captured in bright field mode. The edges of individual cells were traced by hand, and the area enclosed by the trace was measured using Scion Image software (Scion Corp.). Each data point represents a mean of at least 100 individual area measurements. Immunofluorescence analysis was performed as reported in Ref. 14Mancini R. Piccolo E. Mariggiò S. Filippi B.M. Iurisci C. Pertile P. Berrie C.P. Corda D. Mol. Biol. Cell. 2003; 14: 503-515Crossref PubMed Scopus (23) Google Scholar.Analysis of [3H]Inositol-containing PhospholipidsHEK293T and CHO cells were grown in 6-well plates and transfected, and 4 h after transfection they were labeled for 24 h (to isotopic equilibrium) in Medium 199, with 5% FCS containing myo-[3H]inositol (5 μCi/ml). Following labeling, the cells were washed twice with HBSS++ and preincubated for 15 min in HBSS++ containing 10 mm LiCl (pH 7.4) at 37 °C, prior to addition of ATP, as required. Incubations were terminated by medium aspiration and addition of methanol/1 m HCl (1:1, −20 °C), with extraction by addition of a half volume of chloroform (final, 1:1:0.5). After separation of aqueous and organic extraction phases, the [3H]inositol-labeled water soluble metabolites were separated by anion exchange HPLC on a Partisil 10-SAX column using a non-linear water/1 m ammonium phosphate, pH 3.35 (phosphoric acid) gradient. Radioactivity associated with the 3H-labeled compounds was analyzed by an on-line flow detector (Packard FLO ONE A-525). GroPIns levels are given as percentages of total aqueous 3H-labeled compounds. For additional details see Ref. 22Berrie C.P. Iurisci C. Piccolo E. Bagnati R. Corda D. Methods Enzymol. 2007; 434: 187-232Crossref PubMed Scopus (16) Google Scholar.Alkaline Phosphatase Activity and Mineralization AssayTransfected MC3T3-E1 cells from individual wells of a 24-well plate were washed twice with PBS, scraped into alkaline phosphatase buffer (50 mm Tris-HCl, pH 8.0, 0.1% Triton X-100), and sonicated on ice (Handy Sonic; TOMY Seiko, Japan). Alkaline phosphatase activity was assayed by the phosphatase substrate system for EIA (Kirkegaard and Perry Laboratories, Gaithersburg, MD) using the cell lysate supernatant. Activities were corrected for protein concentrations and expressed as nmol/min/mg protein. Transfected MC3T3-E1 cells cultured in osteogenic medium for 7 days were washed twice with PBS and lysed with saline solution containing 10 mm Tris-HCl, pH 7.8, and 0.2% Triton X-100. Thereafter, 0.5 ml 0.5 n HCl was added to lysates and the mineralized materials were dissolved with gentle overnight shaking. The calcium contents were quantitated by the o-cresolphthalein complexone method with the Calcium C-Test (Wako Pure Chemical Industries). Protein concentrations were measured with a Bio-Rad kit.Statistical AnalysisThe data are expressed as means ±S.D./±S.E., as specified, of two to four independent experiments, each performed in duplicate. Statistical analysis was by Student's t test.RESULTSGDE3 Is a Glycerophosphoinositol Inositol PhosphodiesteraseWe have previously shown that GDE1 is a GroPIns phosphodiesterase that selectively hydrolyzes GroPIns over GroPCho (4Zheng B. Berrie C.P. Corda D. Farquhar M.G. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 1745-1750Crossref PubMed Scopus (69) Google Scholar). In addition, we cloned the GDE3 protein in osteoblasts (8Yanaka N. Imai Y. Kawai E. Akatsuka H. Wakimoto K. Nogusa Y. Kato N. Chiba H. Kotani E. Omori K. Sakurai N. J. Biol. Chem. 2003; 278: 43595-43602Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), and its alignment with GDE1 and bacterial phosphodiesterases shows conserved amino acids in the catalytic region, such as an arginine believed to be relevant for GP-PDE activity (Fig. 1A, underlined). To investigate the GP-PDE activity of GDE3, HEK293T cells were transiently transfected with cDNAs coding for wild-type GDE3 with a C terminus green fluorescent protein (GFP) tag (GFP-GDE3wt), a GFP-tagged GDE3 with a catalytic domain point mutation (R231A; GFP-GDE3R), or GFP alone (mock-transfected control) (Fig. 1B).Ten micrograms postnuclear protein from HEK293T cells overexpressing GFP-GDE3wt show a GroPIns inositol phosphodiesterase activity, with hydrolysis of GroPIns to Ins1P and glycerol (see "Experimental Procedures," and Fig. 1, C and D). In comparison, following transfection with pEGFP alone and with pEGFP-GDE3R231A, postnuclear cell lysates showed no background GP-PDE activity over the no-lysate control (Fig. 1D, w/o). There was no evidence of a GDE1-like activity (i.e. hydrolysis of GroPIns to inositol and glycerol phosphate) correlated to GFP-GDE3wt overexpression under these experimental conditions.To determine divalent cation requirements for this GDE3 GP-PDE activity, the GFP-GDE3wt lysate was incubated with 10 mm GroPIns (Li+ salt) in the absence and presence of 10 mm MgCl2, and in the presence of 10 mm MgCl2 plus increasing concentrations of CaCl2 (0.1–10 mm). Unlike the enhancement of GDE1 activity with Mg2+ (4Zheng B. Berrie C.P. Corda D. Farquhar M.G. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 1745-1750Crossref PubMed Scopus (69) Google Scholar), this GDE3 activity did not require MgCl2 (data not shown), but instead required addition of millimolar Ca2+ (Fig. 2A). With 5 mm Ca2+, GroPIns hydrolysis by GFP-GDE3wt was essentially linear over 5 h at 37 °C (Fig. 2B), with no activity in the control GFP postnuclear preparation (Fig. 2C). The Eadie-Hofstee plot derived from GroPIns dose-response analysis (Fig. 2D) shows an apparent Km for GDE3 of ∼97.2 mm, and a Vmax of ∼1,900 pmol/mg/min for GroPIns (see "Experimental Procedures").FIGURE 2.Characterization of GDE3 activity on GroPIns. Postnuclear lysates were prepared from HEK293T cells overexpressing GFP-GDE3wt and GFP (see "Experimental Procedures"). A, quantification of GP-PDE activities on 10 mm GroPIns (Li+ salt) for 2 h at 37 °C with 10 μg of postnuclear GFP-GDE3wt protein in the absence (0) and presence of increasing CaCl2 concentrations, as indicated (see "Experimental Procedures"). Black bars, GroPIns (as substrate); white bars, inositol (Ins); gray bars, Ins1P. GP-PDE activity is given as percentages of total [3H]GroPIns counts added (25,000 dpm on HPLC) for each component. The data are means (±S.E.) of three independent experiments, each carried out in duplicate. B and C, time courses of GDE3 activity on GroPIns with 10 μg of postnuclear GFP-GDE3wt (B) and GFP (C) protein. Black line (squares), GroPIns (as substrate); dashed line (circles), inositol (Ins); gray line (triangles), Ins1P. GP-PDE activity is given as percentages of total [3H]GroPIns counts added (25,000 dpm on HPLC) for each component. The data are from a single representative experiment carried out in duplicate (mean ± S.D.), and are representative of three independent experiments. D, log dose-response curve of postnuclear GFP-GDE3wt activity (over the GFP control) as nmol of GroPIns hydrolyzed after 2 h at 37 °C. The data are from a single experiment carried out in duplicate (mean ± S.D.) and are representative of two independent experiments. E, GPIs competition assay of GDE3 activity toward GroPIns. Quantification of GP-PDE activities of 10 μg of GFP-GDE3wt postnuclear protein on 1 mm GroPIns with 10 mm phosphorylated GPIs, as indicated. The assays were stopped either immediately (T0 control) or after a 2-h incubation at 37 °C (2 h), in the absence (−) and presence of 10 mm of the indicated glycerophosphodiesters (see "Experimental Procedures"). The data are means (±S.E.) of three independent experiments, each carried out in duplicate. Black bars, GroPIns (as substrate); white bars, inositol (Ins); gray bars, Ins1P. GP-PDE activity is given as percentages of total [3H]GroPIns counts added (25,000 dpm on HPLC) for each component. *, p < 0.05, compared with GroPIns alone (paired Student's t test).View Large Image Figure ViewerDownload Hi-res image Download (PPT)GDE3 Is Specific for GlycerophosphoinositolsTo determine the specificity of GDE3, 10 mm GroPCho and GroPIns4P were also used as substrates in phosphodiesterase activity assays with the GFP-GDE3wt postnuclear preparation; neither of these glycerophosphodiesters were hydrolyzed by GDE3 under these conditions.The specificity of GDE3 for GroPIns as substrate was then investigated in competition assays, with the GroPIns concentration reduced to 1 mm to allow addition of a 10-fold excess (10 mm) of unlabeled competing glycerophosphodiesters: GroPCho, glycerophosphoethanolamine, and glycerophosphoserine. Under these conditions, GDE3 GroPIns inositol phosphodiesterase activity was not inhibited (supplemental Fig. S1 SF1). We also tested other GPIs in this competition assay of GDE3, and 10 mm GroPIns4P and 10 mm GroPIns 4,5-bisphosphate competed with GroPIns hydrolysis at least in part (45 and 55% inhibition, respectively) (Fig. 2E).Cells Overexpressing GDE3 Do Not Have Modified Intracellular GroPIns LevelsGlycerophosphodiesterase activity was also tested intracellularly with the HEK293T cells overexpressing GFP-GDE3wt, GFP-GDE3R, and GFP: these transfectants showed comparable intracellular levels of GroPIns (0.52 ± 0.08%, 0.52 ± 0.01%, 0.53 ± 0.07% of total aqueous radioactivity, respectively; n = 3; see "Experimental Procedures").Similar results were obtained with CHO cells, where intracellular GroPIns levels can be modulated by hormone stimulation. Here, addition
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